Method of preparing a killed vaccine for oral use

ABSTRACT

A PROCESS FOR THE PROPAGATION OF RABIES VIRUS AND THE PRODUCTION THEREFROM OF A KILLED RABIES VACCINE WHICH IS SUBSTANTIALLY FREE FROM NON-HUMAN, NON-SPECIFIC PROTEIN IN WHICH THE VACCINE CONTAINS PROTEIN FROM THE GROUP CONSISTING OF FRACTION V OF HUMAN PLASMA AND THE MATERIAL OBTAINED FROM THE SUPERNATANT OBTAINED IN THE PRODUCTION OF FRACTION V BY CONCENTRATION, DIALYSIS AND FREEZE-DRYING OF SAID SUPERNATANT, THE NOMENCLATURE &#34;FRACTION V&#34; BEING THE NOMENCLATURE USED IN THT COHN COLD ETHANOL PROCESS FOR FRACTIONATION OF PLASMA. THE KILLED RABIES VACCINE MAY BE POTENTED BY ADJUVANTS TO ENHANCE IMMUNOGENIC PROPERTY. IN THE FOLLOWING DISCLOSURE, THE WORDS NON-SPECIFIC PROTEIN REFER TO ALL PROTEINS OTHER THAN T HE SPECIFIC PROTEIN WHICH IS RELATED TO THE CELL-ADAPTED RABIES VIRUS WHICH IS USED IN THE INOCULATION OF THE TISSUE CULTURE COMPOSITION OF CELLS.

United States Patent Int. Cl. C12k /00 US. Cl. 424-89 2 Claims ABSTRACT OF THE DISCLOSURE A process for the propagation of rabies virus and the production therefrom of a killed rabies vaccine which is substantially free from non-human, non-specific protein in which the vaccine contains protein from the group consisting of fraction V of human plasma and the material obtained from the supernatant obtained in the production of fraction V by concentration, dialysis and freeze-drying of the said supernatant, the nomenclature fraction V being the nomenclature used in tht Cohn cold ethanol process for fractionation of plasma.

The killed rabies vaccine may be potentiated by adjuvants to enhance immunogenic property.

In the following disclosure, the Words non-specific protein refer to all proteins other than the specific protein which is related to the cell-adapted rabies virus which is used in the inoculation of the tissue culture composition of cells.

This application is a continuation-in-part of my application Ser. No. 614,760, filed Jan. 9, 1967, now abandoned.

SUMMARY OF INVENTION The invention relates to a killed rabies vaccine and the method of producing it, the method having the steps of:

(a) inoculating a tissue culture composition of hamster kidney cells with a strain of hamster kidney celladapted rabies virus, said culture composition being substantially free from non-human protein;

(b) incubating said inoculated composition;

(c) recovering the virus-containing fluid portion of the composition; and

(d) inactivating the virus in the fluid to produce the killed rabies vaccine;

the invention herein defined consisting of the steps of adding to the tissue culture composition at step (a) a protein selected from the group consisting of fraction V of human plasma and the material obtained from the supernatant obtained in the production of fraction V by concentration, dialysis and freeze-drying of said supernatant, said nomenclature fraction V being the nomenclature used in the Cohn cold ethanol process for fractionation of plasma.

PREAMBLE TO DISCLOSURE For many years those working in the field of rabies prophylaxis have endeavoured to develop a satisfactory tissue culture rabies vaccine suitable for human use. It was hoped that such preparations as had been proposed would induce a better immune-response than the presently available vaccines, and that they would eliminate the occurrence of post-vaccinal neuro-complications caused occasionally by rabies vaccines containing animal nervous-tissue.

3,769,415 Patented Oct. 30, 1973 It has been shown in recent years that a rabies vaccine can be produced in tissue culture which, in animal experiments, proves to be efiicient in inducing both serological response and protection against challenge with street rabies virus. The ditficulty has been to prepare such a vaccine free of animal serum, the presence of which renders it unsuitable for human use. The elimination of serum from the nutrient during the process of vaccine preparation results in a substantial drop in vaccine potency. Concentration of the vaccine compensated only partially for the loss.

There is increasing evidence in the last few years that viral vaccines can be significantly potentiated by addition of adjuvants. However, vaccines containing animal tissue, and particularly tissue of nervous origin, are unsuitable preparations for protentiation by adjuvants because of possible allergic reactions which may follow their use in immunization of humans. For example, the administration of vaccine containing nervous tissue of foreign origin can cause serious side elfects in the patient.

It is an object of this invention to provide, by tissue culture methods, rabies vaccine which is substantially free of non-human, non-specific protein and which can be safely administered to humans. I found as a result of my early work that the production of such a vaccine could not be accomplished simply by avoiding addition of foreign protein. If one adopts this course, the potency of vaccine is low and efforts to improve the potency by concentration leave much to be desired.

It might be suggested, of course, particularly after reading some of the early literature in this field, that rabies virus could be propagated by methods comparable to those used for the growing of other viruses. Thus, for example, it is known that poliomyelitis virus grows readily in monkey kidney cells. In some circumstances human plasma or fractions thereof have been added to the monkey kidney cell composition to facilitate the growth of the cells and the propagation of the poliomyelities virus. However, it is worthy of note that the above fact does not apply to rabies virus which, unexpectedly, does not multiply in monkey kidney cells even in the presence of plasma or fractions thereof. Thus, in spite of previous knowledge with other cells and other viruses, I was left with the problem of developing a cell culture method for the production of a rabies vaccine.

The rabies vaccine which is produced by my method is one which is suitable for use in human subjects with or without potentiation by adjuvants. These objectives I accomplish in the manner now described.

DISCLOSURE The main object of this invention is to solve the foregoing problem and to provide a killed (inactivated) rabies vaccine suitable for human use and a method of producing the same. The rabies vaccine produced according to my method is substantially free from protein to which human subjects may show allergic response. The vaccine may be used as such or to prepare an adjuvant-containing vaccine or to prepare an adsorbed rabies vaccine such as aluminum phosphate adsorbed vaccine.

Several methods may be employed for producing the killed (inactivated) rabies vaccine. In the following description, the methods disclosed are for the purpose of illustrating the preparation of my vaccine since it will be within the skill of virologists to employ other selective techniques within the ambit of this disclosure and the appended claims.

One starts, of course, with a strain of rabies virus which maybe selected from several strains commonly used or it may be one developed by selective techniques by the virologist. After selecting the rabies virus, the first step is to adapt it for growth in a tissue culture composition of hamster kidney cells.

One method I employ for producing the killed (inactivated) vaccine is as follows.

Kidneys of weanling hamsters are removed aseptically and after chopping are trypsinized in 0.25% trypsin. After washing the dispersed cells are packed by centrifugation and resuspended to contain approximately 350,000 cells per ml. These are grown in 5-litre flasks in a nutrient medium consisting of Hanks balanced salt solution plus 0.5% lactalbumin hydrolystate plus 10% bovine serum. The medium also contains streptomycin, about 200 units per ml. The cell culture is kept at 37 C. and the medium is changed twice a week. The hydrogen ion concentration of the cell culture is maintained at about pH 7.0-7.2. When a monolayer of cells has formed the cell fluid is removed. The monolayer of cells infected by introducing 40 m1. of seed virus diluted to 1:100, the virus being a hamster kidney-cell-adapted fixed rabies virus strain. The 'bottles are kep at the above temperature. About 14 days after infecting the cell culture, I remove the medium, as by decantation, repetitive washing, etc., and replace with a nutrient medium free from non-human serum but containing a fraction of human blood, namely human albumin, in a concentration of about 0.1%. The cultures are then kept at 32-34 C. for about 4 to 5 days. During this time the conditions I have chosen are such that the cells remain alive and continuously release virus into the fluid which surrounds them. (It will be noted that the composition of the fluid which I have added is one which may be injected with safety and without the occurrence of unfavourable allergic response in humans, such as would be the case if I used in my vaccine the fluid which was earlier present on the cells.) The cell monolayers are then removed from the flasks by repeated freezing-thawing and then added to the respective cell fluids. The cell debris is removed by low centrifugation or filtration.

Having collected the virus fluids from which the vaccine will be produced, I now determine the titre of those fluids. I measure the titre of my virus fluid by determining its LD by intercerebral inoculation of 3-week old mice injecting 0.03 ml. of various dilutions per mouse. The lowest range of a virus fluid useful for the preparation of a rabies vaccine contains approximately l l LD per ml. as described above.

Next, I use one or the other of the following steps which "I refer to as steps A and B. In step A, I subject the material to high speed centrifugation at 65,000 Gs for 2 hours or at 27,500 rpm. in a centrifuge such as Spinco type L equipped with rotor type 30. The pellets are resuspended in supernatant fluid about one-tenth of the original volume. Complete inactivation is achieved with $4 formaldehyde at 37 C. for 8 hours. To neutralize free formaldehyde, I then add 1 stoichiometric unit of sodium bisulphite. Finally thimerosal is added in $4 concentration as a preservative. Next I must satisfy myself, by the commonly accepted and prescribed tests for killed rabies vaccine for human use, that the vaccine is both innocuous and potent. The harvested vaccine may be used in this adjuvant-free form as a killed rabies vaccine or it may be used as the essential component in an adjuvant vaccine. 'If I am preparing an adjuvant vaccine, I add for example aluminum phosphate or aluminum hydroxide. A suitable amount is about 1 mg. per ml. of the vaccine. In these circumstances the antigenicity will be enhanced about four or five-fold.

If I use step B, which is my preference, and which concerns the preparation of a vaccine concentrated by adsorption, the following steps are used. Inactivation is first accomplished by the addition of formaldehyde in a concentration of 4 and the material is kept at 37 C. for 8 hours. I then add sodium bisulphite to neutralize the formaldehyde and I add thimerosal as a preservative as in step A. Now I test the material for innocuity. Having satisfied myself that the virus is killed (and otherwise acceptable, i.e. sterile, non-pyrogenic etc.), I proceed as follows: I add an adjuvant such as aluminum phosphate or aluminum hydroxide. The whole emulsion is agitated throughout about 7 days at 4 C. Then I reduce the volume to one-tenth by low speed centrifugation, discarding nine-tenths of the supernatant fluid and resuspending the precipitate in the remaining one-tenth. Now I test the product for potency by accepted means. Subject to the satisfactory completion of recognized tests (sterility, pyrogenicity, etc.) the product is filled into vials and distributed in the form of a killed, adjuvant-containing rabies vaccine for human use.

Still another variation of my invention, and a very useful variation, is one wherein I proceed as in the first example except that I omit the use of human albumin and add a preparation of polymerized glucose which has been hydrolyzed and sold under the trademark name Dextran. (Preparations of this material are produced having various ranges of molecular size such as 50,000 to 200,- 000. Although any of these may be used, I have a preference for a molecular size about 150,000.) In addition to this glucose preparation I also add a fraction of human plasma which is obtained from the alcoholic supernatant remaining after the precipitation of fraction V in the coldethanol plasma fractionation process of Cohn and his associates. For my use this fraction is prepared by removing alcohol by vacuum distillation. The resulting aqueous concentrate is dialysed against running water for about twenty-four hours and then freeze-dried. The material so produced is hereinafter called super V. In my process I add the resulting dry powder to my culture medium to a concentration of about mg. percent.

In another example of my process I use as the replacement medium free of protein other than of human origin a medium described by my colleagues Healy and Parker (-J. Cell Biology, 30, 531-553, 1966) as medium 1415 DSCV and having the composition noted by them. This medium contains among other components a quantity of polymerized glucose which has been hydrolyzed (sold under the name Dextran) and also a quantity of the above described fraction of human plasma obtained as stated in the preceding paragraph.

The foregoing processes are given by way of illustration of the process for producing a killed rabies vaccine free or substantially free from non-specific protein other than human protein or fractions thereof. The virus is inactivated by conventional means for formaldehyde. I may use my vaccine as an adjuvanted vaccine, the use of adjuvants being known by the prior art. My examples show the use of aluminum hydroxide and aluminum phosphate but I may also use other adjuvants including oily adjuvants.

The value of my rabies vaccine, was tested by conventional laboratory methods, before use on humans, and these tests established its potency and immunizing value.

Among the recognized tests which I conduct on my finished rabies vaccine is one to show that the vaccine is a protective one, the test being the well-known Habel test: published in World Health Organization, Laboratory Techniques in Rabies, Monograph Series No. 23, 1966, page 142. A vaccine which passes this test has an accept able potency for immunizing human subjects.

Then, to demonstrate clearly the use and value of my killed rabies vaccine (containing as an adjuvant aluminum phosphate), produced by one of the foregoing methods, in pre-immunization of humans against exposure to rabies my vaccine was administered in various schedules to 67 students in veterinary medicine. The schedules used were as follows:

Group A23 people-3 doses at 2-week intervals Group B-22 people-3 doses at 4-week intervals Group C22 people-2 doses at 8-week intervals Samples of blood were drawn prior to administration of the vaccine and thereafter at the times noted. Neu- 5 6 tralization tests were conducted on the serum of these (d) adding a nutrient medium to said composition blood samples. The dose of vaccine used was 1 ml. at which medium is substantially free from non-human each injection. The results of serum neutralization tests serum and substantially free from human serum and are shown in the accompanying table. adding to the nutrient medium a fraction of human TABLE-RABIES VACCINE OF TISSUE CULTURE ORIGIN IN FEE-EXPOSURE IMMUNIZATION OF HUMANS [Summary of Neutralization Tests] Distribution of subjects according to serum neutralization titres (50% end points), serum dilutions at i which antibody was detected Immunization schedule su l j e c s Serum samples Undiluted 1:51:25 1:26-1:125 1:1261:625

Group A, 3 doses at 1 ml. 2 week intervals 23 Pre-vaccination (0%) 2weeks after 1st dose--- 9 (40% (22%) 3 (14% 1 (4%) 2weeks after 2nd dose..- 19 (83%) 7 (30%) 10 (43%) 2 (9%) 2weeks after 3rd dose..- 22 (96%) 6 (26%) 14 (61%) 2 (9%) Group B, 3 doses at 1 m1. 4 week intervals 22 Fi e-vaccination 0 (0%) 4 weeks after 1st dose 8 (36%) 4 (18%) 4 (18%) 4 weeks after 2nd dose... 21 (96%) 8 (36%) 10 (45%) 3 (14%) tweaks after 3rd dose... 21 (96%) s (36%) s (36%) 5 (23%) Group C, 2 doses at 1 ml. 8 week intervals 22 Pre-vaccination 0 (0%) Sweeks after 1st dose 10 (45%) 6 (28%) 1 3 dweeks after 2nd dose... 21 (96%) 12 55% 7 (32%) 2 (9% It will be noted that prior to administration of the plasma protein selected from the group consisting of vaccine no subject showed evidence of antibodies to rabies. fraction V and super V, After only 1 dose of my vaccine each group showed a (e) incubating the tissue-cell-culture composition, statistically significant conversion (that is, antibodies to (f) recovering the fluid portion of said last-mentioned rabies) compared with pre-vaccination titres. In each Composition and the Virus, group the improvement in titre which followed a second (8) ihactivaithg the Virus in the fluid PortiOIl to dose of vaccine was statistically significant. In groups B produce a killed rabies vaccine, the preceding steps and C it will be noted that only one person showed no being performed and arranged whereby a vaccine, antibodies after the second dose. In group A it will be suitable for human use, is obtained substantially free noted that only 1 person showed no antibodies after the from non-human P and human Serum except third dose. This experiment demonstrates clearly the value for said one of said fraction V and super V. of my rabies vaccine in immunizing human subjects. No In the method of Producing rabies Virus in hamster subject of the test had :an allergic reaction to the vaccine. kidney cells for the preparation of a killed rabies vaccine Workers in this field may have considered the use of 35 immunizing humans, the steps of freeing hamster human sera. Although such use may a id th use of kidney cells inoculated with a strain of hamster kidney non-human protein u h a bovine u I am fid t cell-adapted rabies virus from substantially all human and that some workers may rai obj tio to th use of non-human protein and then adding the inoculated cells human sera from the standpoint of possible transmission to a nutrient e m to W i h is added a f action of of infectious hepatitis. Whether or not I share this view, human Plasma Protein Selected from the group consisting the fact remains that when I add fractions of human 0f fraction V and p r V, a d nu ri nt medium ing blood, such as albumin and the fraction from the prepasubstantially free from Hon-human Protein and human ration of albumin as noted above, rather adding human Protein except for Said one of said fraction V e Super plasma or h n er I id thi ibl difliculty V whereby a killed rabies vaccine suitable for immunizarising from a virus causing infectious hepatitis or homo- 8 humans is Obtainedlogous serum jaundice, for the preparations which I add can be heated prior to use, if desired, by known means References Clted for protecting against the virus of hepatitis, namely by UNITED STATES PATENTS heating for 10 hours at 60 C. Thus, in my invention I have succeeded in producing a new composition of matter for the production of a killed rabies vaccine for human zv t1 1 OTHER REFERENCES a c aim is: L The method of Producing a killed rabies vaccine Bazeley et al.. Proc. Soc. Exptl. Biol. & Med, vol. 87,

pp. 420-424, 1954, 167/78 V-TC. for immumzmg humans, comprising the steps of can J Microbiol vol 6 479484 0 t b r (a) infecting a tissue culture composition of hamster 1960 c 0 e a j a hamster kldney c611 Fenje: Can. J. MicrobioL, vol. 6, pp. 605-609, Decemb? ii iciiibaii ng zaih i ected composition and thereby ber 1960 167/78 producing a fluid portion of said composition con- 2 Cell B101" 539-553 1966 taining human and non-human serium unsuitable for 7 use as ahuman vaccine, RICHARD L. HUFF, Primary Examiner (c) removing said fluid portion from said composition and discarding said fluid portion containing human US. Cl. XR

and non-human serum, --1.4

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3, 769,415 Dated October 30, 1973 Inventor s) Paul FENJE It is certified that error appears in the above-identified patent and that'said Letters Patent are hereby corrected as shown below:

In the heading, amend the title to read as follows: 'METHOD OF PREPARING'AKILLED VACCINE." Column 2, line 16, "protentiation" should read potentiation Column 2, line 27, after "of" insert the Column 3, line 11, "lactalbumin hydrolystate" should read Lactalbumin Hydrolysate Column 3, line 20, "kep" should read kept Column 5, line 43, after "rather" insert than Column v 5, line 61, "serium" should read serum Column 6, line 26, I "inactivaitng" should read inactivating Signed and sealed this 16th day of July 1974.

(SEAL) Attestz v McCOYYM. GIBSON, JR. C. MARSHALL DANN Attesting Officer Commissioner of Patents FORM po'wso USCOMM-DC seam-pea I Y U.S. GOVERNMENT PRINTING OFFICE: 1969 0-366-33L 

